热带雨林土壤真菌18S rRNA基因多样性分析

目的]探讨热带雨林土壤真菌群落结构,阐释微生物与植物之间的互作关系,以期为开发利用热带雨林这一特殊生态环境中丰富的微生物基因资源提供理论依据。方法]运用扩增18S rDNA限制性分析(Amplified 18S ribosomal DNA restriction analysis,ARDRA)和18S rRNA基因序列分析技术,分析了热带雨林土壤真菌物种丰度、优势种群和进化关系等群落结构特征。试验采用PVPP洗涤-CTAB-溶菌酶-蛋白酶K-SDS热处理等方法,提纯微生物总DNA,构建真菌18S rRNA基因克隆文库,对阳性克隆进行ARDRA分析,选取有代表性的克隆测序,利用DOTUR软件将真菌序列按照97%相似性的标准分成若干个可操作分类单元(Operational taxonomic u-nit,OTU)。结果]构建的克隆文库包含21个OTU,其中以子囊菌和担子菌为主,各占克隆文库的64.7%和15.5%。子囊菌中以盘菌为主,占到整个克隆文库的47.4%;担子菌中以伞菌为主,占到整个克隆文库的10.4%。接合菌和壶菌均有少量克隆出现在文库中。首次从海南岛热带雨林土壤中发现牛肝菌。研究显示该环境真菌多样性极其丰富,涵盖了真菌主要门的绝大多数。结论]该研究结果为揭示热带雨林土壤真菌群落结构多样性、挖掘该环境样品的真菌基因资源提供了参考依据。

Diversity Analysis of Soil Fungal Communities in Tropical Rain Forest by 18S rRNA Gene Cloning

Objective] The aim was to discuss the structures of soil fungi communities in tropical rain forest and expound the interaction between microbes and plants,to provide theoretical basis for exploiting and utilizing abundant microbial resources in the specific tropical rain forest ecological environment.Methods] Amplified 18S ribosomal RNA restriction analysis and 18S rRNA gene cloning library were used to study the structural characteristics of soil fungal species abundance,dominant species and phylogenetic analysis in tropical rain forest for the first time.Total tropical rain forest soil DNA was directly extracted and purified by skiving-thawing-lysozyme-proteinase K-SDS heating treatment and treatment of CTAB.Then 18S rRNA gene cloning library was constructed.After amplified 18S ribosomal DNA restriction analysis,representative clones were sequenced.Fungal sequences were defined into operational taxonomic units(OTUs) according to 97% similarity threshold for OTU assignment was performed using the software program DOTUR.Result] Ascomycotina and Basidiomycota were the two dominant subgroups(64.7% and 15.5% of the data set,respectively) with total 21 OTUs.Pezizomycotina were the dominant sub-groups in the Ascomycotina clones with 47.4% of the data set.Agaricales were the dominant sub-groups in the Basidiomycota clones with 10.4% of the data set.Sequences of Zygomycete,Chytridiomycota were in the library.Boletaceae were discovered in these soil samples for the first time.It might be related to appropriate temperature,high humidity and high content of organic matter.Moreover,fungal diversity is extremely rich,covering the main groups of the vast majority of fungi.Conclusion] The study provided reference basis for excavating the fungal genetic resources and revealing the rich diversity of fungal genetic resources.